Hemoglobin, as the natural oxygen transporter component of blood, is an obvious candidate to form the basis of a blood substitute, e.g. as an aqueous solution. Extensive scientific work has been done and reported, on attempts to provide a satisfactory hemoglobin solution to act as a blood substitute. The chemical properties of hemoglobin outside the red blood cells are, however, markedly different from its properties inside the red blood cells, e.g. as regards its oxygen affinity. The need for some form of chemical modification of hemoglobin to render it suitable for use as a blood substitute has long been recognized and has been quite extensively investigated.
It is well known that hemoglobin comprises a tetramer of four sub-units, namely two .alpha. sub-units each having a globin peptide chain and two .beta. sub-units each having a globin peptide chain. The tetramer has a molecular weight of approximately 64 kilodaltons, and each subunit has approximately the same molecular weight. The tetrameric hemoglobin in dilute aqueous solution readily dissociates into .alpha.--.beta. dimers, and even further under some conditions to .alpha.-sub-unit monomers and .beta.-sub-unit monomers. The dimers and monomers have too low a molecular weight for retention in the circulatory system of the body, and are filtered by the kidneys for excretion with the urine. This results in an unacceptably short half life of such a product in the body. Moreover, uncrosslinked hemoglobin induces significant nephrotoxicity, so that there is a need to minimize the concentration of uncrosslinked hemoglobin in the products. The need for chemical bonding between the sub-units to ensure the maintenance of the tetrameric form ("intramolecular crosslinking") has previously been recognized. Also, the linking together of two or more tetrameric units to form hemoglobin oligomers and polymers of molecular weight greater than 64 kilodaltons ("intermolecular crosslinking") has also been recognized as desirable in many instances.
When present in the red blood cells, hemoglobin is bound to a natural ligand, diphosphoglycerate (DPG) at a particular site in the hemoglobin molecule known as the DPG cleft or pocket. When the red blood cell membrane is removed, the DPG dissociates from the hemoglobin, with consequent steric rearrangement of the hemoglobin molecule and consequent undesirable increase in the affinity of the hemoglobin for oxygen. A satisfactory blood substitute based on hemoglobin should be capable of binding, transporting and releasing oxygen largely in the same manner and under the same conditions as hemoglobin present in natural whole blood. This problem has been addressed in the past by covalently attaching DPG-analogs such as pyridoxal-5'-phosphate PLP to hemoglobin to form the basis of a blood substitute.